This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Transthyretin (TTR) is a homotetramer with each subunit consisting of 127 amino acid residues. Amino acid substitutions in monomeric TTR are hypothesized to destabilize the tetramer and cause the TTR to form intermediates that self-associate into amyloid fibrils. Familial transthyretin amyloidosis (ATTR) is associated with the deposition of TTR variants as amyloid fibrils in various organs and tissues. Since the most effective treatment of ATTR is liver transplantation, correct diagnosis is crucial. MALDI peptide mapping, ESI tandem MS, and/or accurate mass measurements have been used to identify TTR variants. Top down analysis of Tranthyretin (TTR) wild type, Val30Met and Val122Ile variants and TTR from deposited fibrils was first performed by nanospray ESI FTMS using Q2 CAD and SORI-CAD on our hybrid qQq FTMS, the QStar qoTOF MS and the LTQ-Orbitrap MS. TTR patient samples bearing Val30Met and Val122Ile variants and recombinant proteins were analyzed. This constitutes a useful clinical application of top down mass spectrometric analysis. The preliminary top down analysis for TTR variant characterization yields extensive sequence information. We have explored conditions to optimize sequence coverage of TTR using Q2 CAD, and are investigating and MS3 approach where the main fragments generated by Q2 CAD, b42 and y85, are isolated in the quadrupole ion trap or FTMS cell and then undergo dissociation to yield sequence information. Fragmentation was induced in the LTQ-Orbitrap using in-source fragmentation, CAD in the LTQ and the C-trap. These multistage approaches were also used for a TTR sample containing a Val30Met variant. Fragmentation of the b42 fragment bearing the variant allowed a more precise location of the variant to positions 19-32 but did not yield data that specified the mutation site. A similar approach was successfully used to characterize a Val122Ile mutation. BUPID topdown software, writted in-house, is being used for the assignment of peaks in these mass spectra. A manuscript including these results will be included in the special issue of IJMS that honors Nobel prize winner John Fenn. New experiments that involve minimal crosslinking of components of the TTR tetramer are ongoing.